Milk fat comprises membrane-coated droplets of neutral lipid, which constitute the predominant source of lipids for survival of the suckling neonate. From the perspective of the dairy industry, they are the basis for the manufacture of butter and essential ingredients in the production of cheese, yogurt, and specialty dairy produce. To provide mechanistic insight into the assembly and secretion of lipid droplets during lactation, we developed novel intravital imaging techniques using transgenic mice, which express fluorescently tagged marker proteins. The number 4 mammary glands were surgically prepared under a deep plane of anesthesia and the exposed glands positioned as a skin flap with intact vascular supply on the stage of a laser-scanning confocal microscope. Lipid droplets were stained by prior exposure of the glands to hydrophobic fluorescent BODIPY (boron-dipyrromethene) dyes and their formation and secretion monitored by time-lapse subcellular microscopy over periods of 1 to 2 h. Droplets were transported to the cell apex by directed (superdiffusive) motion at relatively slow and intermittent rates (0-2 µm/min). Regardless of size, droplets grew by numerous fusion events during transport and as they were budding from the cell enveloped by apical membranes. Surprisingly, droplet secretion was not constitutive but required an injection of oxytocin to induce contraction of the myoepithelium with subsequent release of droplets into luminal spaces. These novel results are discussed in the context of the current paradigm for milk fat synthesis and secretion and as a template for future innovations in the dairy industry.
Keywords: intravital imaging; lipid droplet fusion; milk lipid; oxytocin-mediated secretion.
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